The D2 dopamine receptor (DAR) is central in the etiology and/or therapy of numerous neuropsychiatric disorders. Unfortunately, truly specific drugs for this receptor have been difficult to obtain, primarily due to high conservation of the orthosteric binding site amongst DARs and other GPCRs. Another approach to receptor selectivity is to identify allosteric modulators that bind to less conserved sites on the receptor. We have developed a high throughput-screening (HTS) platform to identify novel small molecule chemotypes for the D2 DAR and define their DAR selectivity. The primary assay utilizes a cell line expressing an inducible D2 DAR coupled to a chimeric Gqi5 protein, thereby linking D2 DAR activation to robust Ca2+ mobilization measured using a fluorescent readout. We have also developed HTS-formatted secondary assays to measure D2 DAR activities and counter-screening assays to determine DAR subtype selectivity. Through the NIH Molecular Libraries Program, a 370,000+ small molecule library was screened to identify allosteric agonists, as well as positive and negative allosteric modulators. 1,500-2,500 hits were obtained from the primary screen for each class of chemotypes. These hit compounds are being evaluated through a triage plan to characterize their activity and receptor selectivity. Tertiary assays will be performed to identify potential allosteric ligands within each pool of chemotypes. All current antipsychotics are D2 dopamine receptor (D2 DAR) antagonists. These drugs are prone to numerous side effects, which can cause serious morbidity and limit compliance. We are interested in discovering functionally selective ligands as a way of separating therapeutic from unwanted side effects. While G-protein-mediated signaling is more commonly studied, D2 DARs also signal through &#946;-arrestins to activate the Akt-GSK3 pathway. This pathway is involved in dopaminergic actions, and Akt-GSK3 signaling is targeted by lithium in treating bipolar disorder. While clinically active antipsychotics have varying actions on G-protein-mediated signaling, all antipsychotics antagonize D2 DAR/&#946;-arrestin-2 signaling. We have developed two high-throughput screening (HTS) assays to detect D2 DAR-&#946;-arrestin-2 interactions. One involves a BRET-based assay where cells are stably transfected with an inducible D2 DAR-RLuc8 donor construct as well as an m-Venus-&#946;-arrestin-2 acceptor construct. The other HTS assay involves &#946;-galactosidase enzyme complementation to measure D2 DAR/&#946;-arrestin 2 interactions. Through the NIH Molecular Libraries Program, a 350,000 compound library will be screened, and hit compounds will be counter-screened in a G-protein mediated assay. Compounds that are selective for &#946;-arrestin signaling will be evaluated further in terms of receptor selectivity and physiological effects. The D1 dopamine receptor (DAR) plays an important role in cognition, reward and reinforcement, learning and memory, and the regulation of movement. D1-selective agents may prove beneficial in the treatment of many neuropsychiatric disorders, including Parkinsons disease. However, direct D1 stimulation produces rapid desensitization and finding a truly D1-selective agonist has proven difficult due to high sequence similarities among the DARs. Another approach to receptor selectivity is to identify allosteric modulators that bind to less conserved sites on the receptor. Currently, we are using a high throughput screening (HTS) approach, which utilizes a cell line expressing the D1 DAR coupled to G15 resulting in a robust Ca2+ signal upon receptor stimulation, to identify novel D1-selective modulators. Through the NIH Molecular Libraries Program, a 370,000+ small molecule library is being screened to identify allosteric agonists, as well as positive and negative allosteric modulators. From this screen, hit molecules from each class will be chosen for further study. Secondary assays, screens on parental cell lines, and counter screens on other DARS will be used to evaluate activity and receptor selectivity of the compounds identified, and to aid in identifying novel D1 allosteric ligands. The D3 dopamine receptor (DAR) subtype plays a crucial role in regulating movement, mood and emotion, as well as reward and reinforcement. Therapeutically, the D3 DAR may be a useful target for the treatment of drug abuse, as well as neurological and neuropsychiatric disorders. One problem of current drugs, which target the orthosteric site of DARs, is that of cross-GPCR reactivity. However, the development of ligands that target allosteric sites on the receptor may lead to highly specific, efficacious drugs with fewer side effects. We are seeking to identify allosteric modulators of the D3 DAR using a highly robust assay for measuring receptor activity in an HTS format. Our primary screen is an enzyme (&#946;-galactosidase) complementation assay that involves agonist-induced D3 DAR recruitment of &#946;-arrestin, which is measured using a luminescent read-out. We have also developed HTS-formatted secondary assays to measure D3 DAR activities and counter-screening assays to determine DAR subtype selectivity. Through the NIH Molecular Libraries Program, a 370,000+ small molecule library will be screened to identify allosteric agonists, as well as positive and negative allosteric modulators. Resulting hit compounds will be evaluated using a screening plan to characterize their activity and receptor selectivity. Tertiary assays will be performed to identify potential allosteric ligands within each pool of chemotypes.